Funds are requested to purchase a commercial non-linear optical microscope (NLOM) system dedicated for use in cell biological imaging experiments. The NLO system will be based on a Zeiss LSM510 microscope equipped with a Coherent titanium-sapphire femtosecond laser. The instrument will be housed at the Center for Biomedical Imaging Technology (CBIT) at the University of Connecticut Health Center. This research center offers use of high level fluorescence microscopy instrumentation and image analysis resources to the research community both at the UCHC and from outside academic institutions and industry through a well established user facility. It is expected that many of the current 30 laboratories and over 55 graduate and postdoctoral students using the facilities at CBIT will employ the capabilities of the NLO microscope in a wide range research programs. Projects from 6 major users with well developed projects at UCHC are described here. All of these require the deep optical sectioning capabilities characteristic of NLOM and benefit from the suitability of NLOM for imaging living specimens. Dr. Leslie Loew will use NLOM to perform optical recording of membrane electrical properties of neuronal cells in acute brain slice preparations. Dr. John Carson will use NLOM for three dimensional visualization of RNA trafficking in oligodendrocytes in intact optic nerve preparations. Dr. Elizabeth Eipper will follow the expression of GFP-tagged proteins involved in regulated secretion in hippocampal or cerebellar slice preparations. Dr. William Mohler will use two photon imaging to visualize and dissect individual cell fusion events in intact C. elegans embryos. Dr. David Papermaster will study photoreceptor development in transgenic Xenopus tadpoles by following GFP tagged proteins in the living tadpole eye using NLOM. Dr. Mark Terasaki will use NLOM to follow nuclear envelope breakdown in living starfish oocytes, combining high spatial and temporal resolution imaging with photobleaching experiments to assess changes in nuclear envelope and nuclear pore structures that are the antecedents of nuclear envelop breakdown.